Project Summary/Abstract The epithelium of the distal lung performs the critical biological function of gas exchange and serves as a barrier to prevent access of deleterious airborne agents into the body. In order to maintain these functions the lung has an extensive ability to respond to injury and regenerate lost or damaged cells. Current models posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, we have recently defined the regenerative role of rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Orthotopic transplantation of LNEPs reveals a striking capacity for direct differentiation into type 2 pneumocytes and distal airway cells. However, after severe influenza injury, LNEPs activate a ?Np63 and cytokeratin 5 (Krt5) remodeling program whereupon cells proliferate and migrate toward heavily injured alveolar areas. This pathway largely results in an abnormal epithelial barrier rather than type II cell differentiation. The dysplastic alveolar epithelial cysts result i part from ongoing excessive Notch signaling and are reminiscent of honeycombing cysts in fibrotic human lungs. Interestingly, transplantation of the total LNEP population demonstrates both bronchiolar and alveolar differentiation, whereas cells expressing low levels of Krt5 are heavily biased toward airway-like cystic expansion. Furthermore, principle component analysis of single cell transcriptomes indicates multiple subpopulations of cells. These observations suggest that heterogeneity within the LNEP population is a manifestation of lineage-primed subpopulations predisposed towards particular fates. This proposal seeks to address the biological significance of this heterogeneity with the following specific aims: 1) Test whether murine LNEPs consist of both unbiased and primed stem/progenitor cell populations. 2) Identify the human equivalent(s) of LNEPs and explore their role in human lung disease. 3) Define distinct signaling cues that modulate airway and alveolar regenerative responses of unbiased and primed LNEP subpopulations. These aims will utilize single cell RNA-Seq, lineage tracing, organoid culture, and orthotopic cell transplantation to interrogate cellular heterogeneity and regenerative dynamics with a high degree of tractability. These studies will lay the foundation for development of a quantitative and predictive model of lung epithelial injury responses. Such a model will inform future efforts to manipulate regenerative mechanisms in order to achieve better disease outcomes.